Chimeric bacteriophage lysin with activity against staphylococci bacteria

ABSTRACT

The present disclosure relates to chimeric bacteriophage lysins useful for the identification and/or reduction of staphylococcal populations. For example, a chimeric bacteriophage lysin was engineered and shown to effectively kill all strains of staphylococci tested including antibiotic resistant methicillin-resistant S. Aureus and VISA.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/078,277 filed on Jul. 3, 2008, which is hereinincorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under grant numberA111822 awarded by the National Institutes of Health (NIH). The U.S.government may retain certain rights to the invention.

TECHNICAL FIELD

The present disclosure relates to the identification and use of chimericlytic enzymes to rapidly and specifically detect and kill Staphylococcibacteria, including certain antibiotic-resistant Staphylococcus aureusbacterial strains.

BACKGROUND

Staphylococcus aureus is an opportunistic pathogen inhabiting human skinand mucous membranes. S. aureus is the causative agent of variety ofskin and soft tissue infections in humans and serious infections such aspneumonia, meningitis, endocarditis, and osteomyelitis. S. aureusexotoxins also cause disease syndromes such as bullous impetigo, scaldedskin syndrome, and toxic shock syndrome. Additionally, staphylococci arealso among the most common causes of food-borne illness in United States(Fischetti V A, Novick, R. P., Ferretti, J. J., Portnoy, D. A. and Rood,J. I., editor. 2006. Gram-positive pathogens. 2nd ed: ASM Press). S.aureus is also a major cause of community- and hospital-acquired(nosocomial) infections. Of the nearly 2 million cases of nosocomialinfections in United States, approximately 230,000 cases are caused byS. aureus (NNIS. 2003. NNIS report, data summary from January 1992through June 2003, issued August 2003. American Journal of InfectionControl 31:481-498.).

The global appearance of methicillin- and vancomycin-resistant clinicalisolates of S. aureus has become a serious concern. Currently, 40-60% ofnosocomial infections of S. aureus are resistant to oxacillin (Massey RC, Horsburgh M J, Lina G, Hook M, Recker M. 2006. The evolution andmaintenance of virulence in Staphylococcus aureus: a role forhost-to-host transmission? Nat Rev Microbiol 4(12):953-8.) and greaterthan 60% of the isolates are resistant to methicillin (Gill S R, Fouts DE, Archer G L, Mongodin E F, Deboy R T, Ravel J, Paulsen I T, Kolonay JF, Brinkac L, Beanan M and others. 2005. Insights on evolution ofvirulence and resistance from the complete genome analysis of an earlymethicillin-resistant Staphylococcus aureus strain and abiofilm-producing methicillin-resistant Staphylococcus epidermidisstrain. J Bacteriol 187(7):2426-38.). Treating infections caused by thedrug-resistant S. aureus has become increasingly difficult and thereforeis a major concern among healthcare professionals. To combat thischallenge, development of new and effective antibiotics belonging todifferent classes are being aggressively pursued. A number of newantimicrobial agents such as linezolid, quinupristing-dalfopristin,daptomycin, tigecyline, new glycopeptides and ceftobiprole have beenintroduced or are under clinical development (Aksoy D Y, Unal S. 2008.New antimicrobial agents for the treatment of Gram-positive bacterialinfections. Clin Microbiol Infect 14(5):411-20.). However, clinicalisolates of MRSA (methicillin-resistant Staphylococcus aureus) withresistance to these new classes of antibiotics have already beenreported (Tsiodras S, Gold H S, Sakoulas G, Eliopoulos G M, WennerstenC, Venkataraman L, Moellering R C, Ferraro M J. 2001. Linezolidresistance in a clinical isolate of Staphylococcus aureus. Lancet358(9277):207-8; Mangili A, Bica I, Snydman D R, Hamer D H. 2005.Daptomycin-resistant, methicillin-resistant Staphylococcus aureusbacteremia. Clin Infect Dis 40(7):1058-60; Skiest D J. 2006. Treatmentfailure resulting from resistance of Staphylococcus aureus todaptomycin. J Clin Microbiol 44(2):655-6). Consequently, there is anurgent need to develop novel therapeutic agents or antibioticalternatives against MRSA.

Bacteriophage endolysins (lysins) are one such class of novelantimicrobial agents that are emerging as novel agents for theprophylactic and therapeutic treatment of bacterial infections. Lysinsare cell wall hydrolases that are produced during the infection cycle ofdouble-stranded DNA bacteriophages (or phages) enabling release ofprogeny virions. Typically, lysins have two distinct functional domainsconsisting of a catalytic domain for peptidoglycan hydrolysis and abinding domain for recognition of surface moieties on the bacterial cellwalls. The catalytic domains are relatively conserved among lysins. Theactivities of lysins can be classified into two groups based on bondspecificity within the peptidoglycan: glycosidases that hydrolyzelinkages within the aminosugar moieties and amidases that hydrolyzeamide bonds of cross-linking stem peptides. The binding domains howeverare not conserved among lysins. Hence the binding domain impartsspecies- and strain-specificity because the binding targets, oftencarbohydrates associated with the peptidoglycan, display species- orstrain-specific distribution (Fischetti V A, Nelson D, Schuch R. 2006.Reinventing phage therapy: are the parts greater than the sum? NatBiotechnol 24(12):1508-11). The modular architecture of lysins' is animportant feature with respect to their development as antimicrobialagents. This enables creation of chimeras by swapping lysin domains andthereby altering binding specificity or enzymatic activity or both(Sheehan M M, Garcia J L, Lopez R, Garcia P. 1996. Analysis of thecatalytic domain of the lysin of the lactococcal bacteriophage Tuc2009by chimeric gene assembling. FEMS Microbiol Lett 140(1):23-8; Lopez R GE, Garcia P, Garcia J L. 1997. The pneumococcal cell wall degradingenzymes: a modular design to create new lysins? Microb Drug Res3:199-211; Croux C, Ronda C, Lopez R, Garcia J L. 1993. Interchange offunctional domains switches enzyme specificity: construction of achimeric pneumococcal-clostridial cell wall lytic enzyme. Mol Microbiol9(5):1019-25; Donovan D M, Dong S, Garrett W, Rousseau G M, Moineau S,Pritchard D G. 2006. Peptidoglycan hydrolase fusions maintain theirparental specificities. Appl Environ Microbiol 72(4):2988-96).

When applied exogenously, native or recombinant lysins were able todegrade the cell wall of susceptible bacteria and cause rapid cell lysis(Nelson D, Loomis L, Fischetti V A. 2001. Prevention and elimination ofupper respiratory colonization of mice by group A streptococci by usinga bacteriophage lytic enzyme. Proc Natl Acad Sci USA 98(7):4107-12).Lysins have been developed against a number of Gram-positive pathogensincluding Group A streptococci (Nelson D, Loomis L, Fischetti V A. 2001.Prevention and elimination of upper respiratory colonization of mice bygroup A streptococci by using a bacteriophage lytic enzyme. Proc NatlAcad Sci USA 98(7):4107-12), S. pneumoniae (Loeffler J M, Nelson D,Fischetti V A. 2001. Rapid killing of Streptococcus pneumoniae with abacteriophage cell wall hydrolase. Science 294(5549):2170-2), Bacillusanthracis (Schuch R, Nelson D, Fischetti V A. 2002. A bacteriolyticagent that detects and kills Bacillus anthracis. Nature418(6900):884-9), enterococci (Yoong P, Schuch R, Nelson D, Fischetti VA. 2004. Identification of a broadly active phage lytic enzyme withlethal activity against antibiotic-resistant Enterococcus faecalis andEnterococcus faecium. J Bacteriol 186(14):4808-12), Group B streptococci(Cheng Q, Nelson D, Zhu S, Fischetti V A. 2005. Removal of group Bstreptococci colonizing the vagina and oropharynx of mice with abacteriophage lytic enzyme. Antimicrob Agents Chemother 49(1):111-7),and Staphylococcus aureus (Rashel M, Uchiyama J, Ujihara T, Uehara Y,Kuramoto S, Sugihara S, Yagyu K, Muraoka A, Sugai M, Hiramatsu K andothers. 2007. Efficient elimination of multidrug-resistantStaphylococcus aureus by cloned lysin derived from bacteriophage phiMR11. J Infect Dis 196(8):1237-47). The activities of most of theselysins have been demonstrated in vitro and in in vivo models. Severalunique characteristics of lysin make them attractive antibacterialcandidates against Gram-positive pathogens. These include i) rapidantibacterial activity both in vitro and in vivo; ii) very narrow lyticspectrum (species- and strain-specific); iii) very strong bindingaffinity, typically in the nanomolar range; iv) very low chances ofdeveloping resistance since the binding epitopes are essential forviability; v) safe; and vi) relative ease of modification by geneticengineering (Fischetti V A, Nelson D, Schuch R. 2006. Reinventing phagetherapy: are the parts greater than the sum? Nat Biotechnol24(12):1508-11).

Although lysins have been developed against a number of Gram-positivepathogens, there remains a need for a S. aureus-specific lysin. Variouslabs have unsuccessfully attempted to obtain a staphylococcal lysin. Theexpression of more than twenty different staphylococcal lysins using avariety of techniques have been attempted without success. These includeexpression of lysin genes in E. coli using different expression vectorsand conditions, expression in Bacillus, yeast and mammalian systems,expression in the presence of chaperones, expression of truncatedversions etc. To our knowledge, there is only one report of thesuccessful development of S. aureus-specific lysin called MV-L (RashelM, Uchiyama J, Ujihara T, Uehara Y, Kuramoto S, Sugihara S, Yagyu K,Muraoka A, Sugai M, Hiramatsu K and others. 2007. Efficient eliminationof multidrug-resistant Staphylococcus aureus by cloned lysin derivedfrom bacteriophage phi MR11. J Infect Dis 196(8):1237-47). MV-L lysin iscomprised of two catalytic domains (an endopeptidase and an amidasedomain) linked to a single cell wall targeting (CWT) domain, a type ofbinding domain. Unless otherwise indicated, references herein to a“binding domain” herein include a CWT domain. The MV-L CWT domain, likethe staphylolytic enzyme lysostaphin, displays homology to SH3b-likedomains. The SH3b-like domains bind to the peptide cross-bridge (thepenta Glycine) in the staphylococcal cell wall. There are reports ofstaphylococcal strains developing resistance at 10⁻⁶ frequencies tolysostaphin by altering their peptide cross-bridges. Therefore, weexpect staphylococci to develop resistance at a higher frequency tolysins containing SH3b-like CWT domains including MV-L. There is a needfor lytic enzymes capable of specific binding to Staphylococcal bacteriawithout undesirably high frequencies of lysostaphin resistance, such asS. aureus—specific lysins without SH3b-like CWT domains.

SUMMARY

This disclosure describes novel staphylococcal lysins, as well asmethods of making and using the lysin. In one example, the geneticengineering of a novel chimeric lysin called ClyS (for chimeric lysinfor staphylococci) is described. ClyS is specifically active againstsusceptible and drug-resistant staphylococci, and was constructed byfusing the catalytic domain of a Staphylococcus-specific phage lysinwith a unique binding domain from another Staphylococcus-specific phagelysin that has no known homologs. ClyS is a solubleStaphylococcal-specific lysin without a SH3b-like CWT domain, but doescontain a CWT domain that is believed to recognize astaphylococci-specific surface carbohydrate. Consequently, the frequencyby which staphylococcal strains will develop resistance to ClyS may bereduced. Additionally, biochemical characterization of ClyS revealedthat the pH and salt spectrum of ClyS is very different fromconventional lysins thereby providing unique properties to this chimericlysin.

Also included within the scope of the present invention are methods ofusing the binding domain for diagnostic purposes, the method comprisingthe steps of contacting a sample with a reporter molecule comprising acell wall target domain comprising the amino acid sequence of SEQ IDNO:1 and a fluorescent reporting moiety bound thereto; and subsequentlydetecting the presence of the reporter molecule bound to astaphylococcus bacteria within the sample. In certain embodiments, thereporter molecule is a green fluorescent protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of phiNM3 lysin showing the putative CHAP(“cysteine- and histidine-dependent amidohydrolase/peptidase”) and CWTdomains. The numbers represent the amino acid positions and the domainlimits. The CWT domain of ClyS is indicated in the diagram.

FIG. 2A is a gel showing the purification of phiNM3 CWT. SDS-PAGE andcoomassie blue stained gel of phiNM3 CWT purified by anion-exchangechromatography is depicted in the lane marked “CWT.” Protein molecularweight markers in kilodaltons (kDa) are shown in the lane marked “M.”

FIG. 2B shows the amino acid sequence of the phiNM3 CWT protein (SEQ IDNO:1).

FIG. 3 shows a series of micrographs showing PhiNM3 CWT bindingspecifically to staphylococci. Purified phiNM3 CWT was labeled with FITCand exposed to 1) S. aureus; 2) B. cereus; 3) S. epidermidis; 4) E.coli; 5) Group A Streptococcus and 6) mixed suspension of S. aureus andB. cereus cells. “P” indicates phase-contrast image and “F” indicatesfluorescent image.

FIG. 4 is a schematic diagram illustrating chimeric lysin development.In particular, FIG. 4 provides schematic diagrams of various chimericlysins showing their respective domains and the corresponding expressionand solubility of the protein and activity against S. aureus cells.Similar domains are depicted in the same shading and labeled. PlyB-catindicates catalytic domain of Bacillus-specific lysin PlyB (and ismarked with a “4” in the figure); Sa-aa indicates 16 amino acid residuesspecific for staphylococcal lysins (and is marked with a “5” in thefigure); PlyB-CWT indicates CWT domain of PlyB (and is marked with a “6”in the figure); Twort-CWT indicates CWT domain of S. aureus phage Twortlysin (and is marked with a “8” in the figure); Lysostaphin CWtindicates CWT domain of lysostaphin (and is marked with a “10” in thefigure); and Se autolyin amidase indicates an amidase domain of S.epidermidis autolysin (and is marked with a “12” in the figure).

FIG. 5A shows the ClyS protein sequence. The predicted protein sequenceof the chimeric protein ClyS showing the Twort endopeptidase catalyticand the phiNM3 CWT domains.

FIG. 5B shows the amino acid sequence for the AD127 chimeric molecule,described with respect to FIG. 4.

FIG. 5C shows the amino acid sequence for the native (unmodified) Twortlysin (SEQ ID NO: 12).

FIG. 6 is a gel showing the purification of ClyS. ClyS was expressed inE. coli DH5α cells and purified by cation-exchange chromatographyfollowed by hydroxyapatite chromatography. Purified sample (10micrograms) was separated by SDS-PAGE and stained by Coomassie blue(right hand lane). Protein molecular weight markers in kilodaltons (kDa)are shown in the left hand lane.

FIG. 7 is a graph showing the activity of ClyS against S. aureus invitro. S. aureus strain 8325-4 cells were resuspended in 20 mM phosphatebuffer (pH 7.4), incubated with 50U of ClyS and OD600 (filled triangles)monitored by a spectrophotometer. Control experiments (filled squares)were performed under the same conditions with buffer alone. Viability(filled diamonds) of cells, shown as colony-forming units/ml, wasdetermined by serially diluting and plating the cells.

FIG. 8 is a series of micrographs showing that ClyS causes cell walldisruption and ultimately lysis of 8325-4 cells. FIGS. 8A-8C (A-C) arethin-section transmission electron micrographs (bars, 200 nm) of S.aureus 3 minutes after exposure to 50 U of ClyS. The arrows indicatecytoplasmic membrane extrusions through holes generated in the cell wallby ClyS. Ultimate lysis results in “cell-ghosts” (D) after the loss ofcytoplasmic contents (bar, 500 nm).

FIGS. 9A and 9B are graphs showing the activity of ClyS in various pHand salt concentration conditions. FIG. 9A is a graph of the activity ofClyS (50U) tested against S. aureus strain 8325-4 in buffers with pHvalues ranging from 4 and 10 in 15 minute assays. Optical density(filled squares) and viability (filled diamonds) was measured asdescribed in legend of FIG. 6. Fold killing in the viability assay wascalculated by dividing the number of viable bacteria after buffertreatment at a particular pH by the number after exposure to ClyS enzymeat the same pH. Final pH readings for each reaction are recorded on thex axis. FIG. 9B is a graph showing the activity of ClyS (50 U) testedagainst S. aureus strain 8325-4 in 20 mM phosphate buffer (pH 7.4) inthe presence of different concentrations of NaCl. After 15 minutessamples were assayed for optical density and viability calculated asabove.

FIG. 10 is a bar graph showing that ClyS exerts specific killing ofstaphylococci. Log-phase cultures of different bacteria were exposed to50 U of ClyS for 15 minutes. Fold killing was calculated as described inFIG. 8 legend.

FIG. 11 depicts a graph of the CFU of MRSA from individual MRSA infectedmice after being administered phosphate buffered saline pH 7.3 (control)or ClyS (630 μg).

FIG. 12 depicts Kaplan Meier Survival Curves showing the effect of ClySon preventing death in mice injected with MRSA compared with phosphatebuffer control.

FIG. 13 depicts an isobologram for a checkerboard broth microdilutionstudy of the effect of vancomycin on VISA (vancomycin-resistantStaphylococcus aureus) or oxacillin on MRSA with increasing amounts ofClyS.

FIG. 14 depicts Kaplan Meier Survival Curves showing the effect ofoxacillin alone or in combination with ClyS.

FIG. 15 depicts a photograph of a Coomassie-blue stained SDS-PAGE gel ofa 5-day time-course at 21° C. of ClyS in the absence (top left gel) orpresence of 5 mM DTT (top right gel), and pClyS in the absence (bottomleft gel) or presence of 5 mM DTT (bottom right gel). About 20micrograms of protein was loaded into each lane of the gel. The bottomright gel shows a much higher amount of intact pClyS in the presence of5 mM DTT after 5 days compared to intact ClyS in the presence of 5 mMDTT after 5 days.

DETAILED DESCRIPTION Definitions

Unless otherwise indicated, the certain terms used herein and theirapplicability to the present disclosure are defined below.

The term “isolated” means at least partially purified from a startingmaterial. The term “purified” means that the biological material hasbeen measurably increased in concentration by any purification process,including by not limited to, column chromatography, HPLC, precipitation,electrophoresis, etc., thereby partially, substantially or completelyremoving impurities such as precursors or other chemicals involved inpreparing the material. Hence, material that is homogenous orsubstantially homogenous (e.g., yields a single protein signal in aseparation procedure such as electrophoresis or chromatography) isincluded within the meanings of isolated and purified. Skilled artisanswill appreciate that the amount of purification necessary will dependupon the use of the material. For example, compositions intended foradministration to humans ordinarily must be highly purified inaccordance with regulatory standards.

The term “lytic enzyme genetically coded for by a bacteriophage” refersto a polypeptide having at least some lytic activity against the hostbacteria.

Variants of “chimeric bacteriophage lysin” are included within thedefinition of chimeric bacteriophage lysins, and include a functionallyactive chimeric bacteriophage lysin with killing activity againstStaphylococcus aureus having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or even at least 99.5% amino acid sequenceidentity with a sequence described herein. For example, the presentinvention includes chimerical bacteriophage lysins having at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or even atleast 99.5% amino acid sequence identity with the polypeptide sequenceof SEQ ID NO:2.

“Percent (%) polypeptide sequence identity” with respect to the lyticenzyme polypeptide sequences identified here is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the specific lytic enzymepolypeptide sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Methods for alignment for purposes of determining percentamino acid sequence identity are described below.

Staphylococcal Lysins

Chimeric bacteriophage lysins with killing activity against S. aureusare described herein. Lysins generally occur in a modular structure.FIG. 1 is a schematic diagram of phiNM3 lysin showing the putative CHAPdomain 110 and the CWT domain 120. The numbers represent the amino acidpositions and the domain limits. The CWT domain of ClyS is shown asshaded box 120. The N-terminal module consists of a catalytic domainbelieved to possess the ability to break down the bacterial cell wall ofcertain bacteria. Enzymatic activities often associated with thecatalytic domain are amidases, endopeptidases, glucosamidases andmuramidases. The C-terminal module consists of a binding domain that isbelieved to have an affinity for a carbohydrate epitope on the targetbacteria cell wall. The binding domain is believed to determine thespecificity of the lysin. The peptide cross-bridge within thestaphylococcal peptidoglycan is believed to function as the receptor forthe CWT domain of lysostaphin, a staphylolytic enzyme produced byStaphylococcus simulans. The CWT domain of lysostaphin has homology tothe SH3b domain suggesting that such lysins might also utilize thepeptide cross-bridge as its receptor.

In one embodiment, Staphylococcus-specific binding molecules comprisinga CWT domain within staphylococcal lysins are provided that have noknown domain homologs. In some embodiments, the binding molecules arelysins. In other embodiments, the binding molecules may be used asdiagnostic tools, for example to identify the presence of Staphylococcusbacteria. Preferably, such a CWT domain is provided to recognize adifferent epitope such as a cell wall-associated carbohydrate instead ofthe peptide cross-bridge in the staphylococcal cell wall.

In a further embodiment, the ClyS lysine can be used to digest the cellwall of Staphylococcus aureus bacterial strains, which in turn wouldallow access to the genetic and cytoplasmic material, such as endogenousDNA and RNA, to further identify and sequence the Staphylococcus aureusbacterial strain. It will also release membrane-associated andwall-associated molecules for diagnostic purposes.

Most preferably, the binding molecule is a soluble binding domain of abacterial lysin comprising a polypeptide including an amino acidsequence providing specific binding to S. aureus, such as SEQ ID NO:1(phiNM3 CWT domain). For example, the lysin preferably includes thepolypeptide sequence of S. aureus phage phiNM3 lysin (SEQ ID NO:1)(protein accession number YP_908849). The phiNM3 lysin CWT domain (SEQID NO:1) corresponding to amino acid residues 158-251 was cloned andexpressed. The approximately 10-kDa protein of SEQ ID NO:1 was highlysoluble and was purified by one-step anion-exchange chromatography tohomogeneity. FIG. 2A is an anion exchange gel showing the protein of SEQID NO:1 in a second column next to a set of marker proteins in a firstcolumn. FIG. 2B shows the amino acid sequence of SEQ ID NO:1. Todetermine whether the peptide domain of SEQ ID NO:1 displayedStaphylococcus-specific binding, the purified protein was labeled withFITC and exposed to log-phase S. aureus, S. epidermidis and mixedpopulation of S. aureus and Bacillus. Group A streptococci, E. coli andBacillus cereus were used as controls. More preferably, The FITC-labeledphiNM3 CWT domain bound specifically to S. aureus (FIG. 3-1) and S.epidermidis (FIG. 3-3) cells when present in single or mixed populations(FIG. 3-6) while binding to streptococci (FIG. 3-5), Bacillus (FIG. 3-2)or E. coli (FIG. 3-4) was not observed. PhiNM3 lysin specifically boundto S. aureus (FIG. 3-1) and S. epidermidis (FIG. 3-2) cells when presentin single or mixed populations (FIG. 3-3) while binding to streptococci(FIG. 3-4), Bacillus (FIG. 3-5) or E. coli (FIG. 3-6) was not observed.

In one embodiment, the binding molecule comprises a CWT binding domain,such as the amino acid sequence of SEQ ID NO:1, attached to a reportingportion that is detectable to identify the presence of the bindingmolecule bound to Staphylococcal bacteria. For example, the bindingmolecule may include the amino acid sequence of SEQ ID NO:1 bound to afluorescent reporter group, a radioactive reporter group or aheterologous tag that is adapted to bind a fluorescent reporter. ThephiNM3 (SEQ ID NO:1) CWT domain may be used as a diagnostic tool for theidentification of staphylococcal bacteria. The high affinity bindingsite may be used in a wide range of assay techniques to detect S.aureus. Such assay methods include radioimmunoassays, gold sol radialimmune assays, competitive-binding assays, Western Blot assays and ELISAassays. Such detection assays advantageously utilize a heterogeneousformat wherein a binding reaction (SEQ ID NO:1) between a conjugatedbinding agent comprising (SEQ ID NO:1) and an analyte occurs followed bya wash step to remove unbound conjugated binding agent. For example,gold sol particles may be prepared with protein that comprises thebinding region with the binding protein immobilized on the particlesurfaces. As binding occurs between the protein and (staphylococcal)bacteria, the particles merge and form a colored product. Analogously,the binding protein may be complexed, preferably covalently with anenzyme such as beta galactosidase, peroxidase, or horseradishperoxidase. After wash, the remaining bound enzyme can be detected byadding a substrate such as a fluorogenic or chemilumigenic substrate.The binding protein may be complexed with any other reagent that canmake a signal such as a rare earth fluor and detected by time resolvedfluorescence, a radioactive material and detected by radioactivitymeasurement, green fluorescent protein (GFP) or another fluorescent tag,and detected by fluorescence.

For comparison, FIG. 5B provides the amino acid sequence of SEQ ID NO:3,the AD119 sample discussed with respect to FIG. 4. AD119 (SEQ ID NO:3)comprises the Twort endopeptidase domain joined to the Lysostaphin CWTdomain. In contrast to the chimeric compound of SEQ ID NO:2 (AD127),which shares the Twort endopeptidase domain but has the phiNM3 CWTdomain (SEQ ID NO:1) in place of the Lysostaphin CWT domain, the AD127compound was insoluble and exhibited little or no killing activityagainst S. aureus.

The conjugation of the binding region with a detectable tag may becarried out by synthetic chemistry or a biological process. For example,a DNA sequence coding for the binding region of SEQ ID NO:1 or of theentire lysin of SEQ ID NO:2 can be linked to genetic information thatencodes a detectable marker such as green fluorescent protein (GFP) oran enzyme such as alkaline phosphatase. This could be accomplished byseparating the DNA for the binding domain by removing the N-terminalcatalytic domain and replacing it in frame with indicator molecules suchas green flourescent protein (GFP) and purifying the expressed fusionmolecule for the identification of S. aureus. Since the binding domainhas a similar binding affinity of an immunoglobulin G molecule, themarked binding domain will effectively identify Staphylococcus aureuswith little false positive activity. One also could fuse the GFPmolecule or an enzyme at the 5′ end of the whole lysin enzyme ifnecessary, by doing so the enzymatic domain will be at least partlyinactivated, still allowing the binding domain to function to bind toits substrate in the bacillus cell wall. Optionally, the isolatedbinding domain of SEQ ID NO:1 may be separated from the catalytic domainof SEQ ID NO:2 and may be expressed, purified and labeled using a numberof fluorescent molecules such as fluorescein isothiocyanate, rhodamineisothiocyanate and others known by skilled artisans. The binding domainmay be modified with biotin to allow formation of a biotin-avidincomplex after the binding region adheres to the Staphylococcus aureusfor identification.

In another embodiment, the lysin is a chimeric protein that comprises anendopeptidase domain of the S. aureus Twort lysin upstream of the phiNM3CWT domain (SEQ ID NO:1). The chimeric polypeptide is preferablysufficiently soluble in phosphate buffered saline (PBS). Preferredlevels of solubility in PBS for the chimeric lysins is at least about 1mg/ml and more preferably at least about 3 mg/mL in PBS. While nativestaphylococcal bacteriophage lysins are typically insoluble in PBS, thechimeric lysins comprising an endopeptidase domain of a first lysin(e.g., Twort S. aureus lysin) bound to the CWT domain of SEQ ID NO:1 aresurprisingly soluble in PBS (e.g., at least about 1 mg/ml, and typicallyabout 3 mg/ml or greater). One example of such a lysin is provided inSEQ ID NO:2 (AD127), shown in FIG. 5A and consisting of the Twort lysinendopeptidase domain attached to the phiNM3 CWT domain (SEQ ID NO:1).The isolated polypeptide of SEQ ID NO:2 (AD 127) was constructed byengineering S. epidermidis autolysin amidase and Twort lysinendopeptidase domains upstream of phiNM3 CWT domain, respectively.Chimera AD 126 had no expression or activity but AD 127 was soluble andhad very high activity but low expression. To overcome low expression ofAD 127 construct, the entire chimera gene was cloned into expressionvector pJML6 to generate pAD 138. The expression, solubility andactivity of AD 127 from the pAD138 construct was very high. Therefore,this chimera was named ‘ClyS’ for Chimeric lysin for Staphylococcus(FIG. 5A).

ClyS (SEQ ID NO:2) contains 280 amino acid residues with a deducedmolecular mass of 31956 Da and a theoretical isoelectric point of 9.17,and was purified by two-step column chromatography to >90% homogeneity.ClyS had a molecular mass of approximately 31 kDa by SDS/PAGE (FIG. 6)which was confirmed by gel filtrations chromatography, suggesting thatthe protein exists as a monomer and is not proteolytically processed(data not shown).

The unit activity of ClyS was defined by measuring thespectrophotometric loss of turbidity, indicative of cell lysis, of S.aureus 8325-4 cells upon adding serial dilutions of ClyS. In our assays,5 micrograms of ClyS corresponded to 1 U of lytic activity. When 50 U ofClyS was added to exponentially growing 8325-4 cells the OD600 droppedto baseline within 5 min (FIG. 7). To confirm that the observed celllysis corresponds to cell death, staphylococcal viability was determinedby enumerating aliquots from the lytic reaction at various time points.A decrease in viability of approximately 3-logs was observed in 30 min(FIG. 7).

The lytic effect on S. aureus 8325-4 cells exposed to 50U of ClyS for1-3 min was visualized by transmission electron microscopy. Typical oflysin activity observed previously, localized degradation of the cellwall was observed at single (FIG. 8A) or multiple sites (FIG. 8B).However, unlike other lysins, the sites of degradation on the cell wasnot restricted to the septal or polar positions but was randomlydistributed. This resulted in extrusions and rupture of the cellmembrane (FIG. 8C) and subsequent loss of cytoplasmic contents andformation of cell-ghosts (FIG. 8D).

The effect of pH on the activity of ClyS was determined by measuring thedrop in OD₆₀₀ or cell viability at different pH values. We observed thatClyS was active over a wide range of pH values but was most activebetween pH 9 and 10. However, ClyS retained partial yet significantactivity at physiological pH (FIG. 9A). Similarly, the effect of saltconcentration on activity of ClyS was also determined. ClyS displayedactivity in a wide range of salt concentrations (FIG. 9B). While itsactivity deteriorated above 400 mM NaCl, at physiological concentrationsClyS functioned well.

Muralytic activity of ClyS was tested on a number of bacterial strainsrepresenting a variety of species which were divided into sets (Table 1and FIG. 10). Set I consisted of S. aureus strains includingmethicillin-sensitive S. aureus (MSSA) and MRSA. ClyS was active againstMSSA and MRSA although differences were observed between S. aureusstrains. Set II consisted of different species of staphylococciincluding S. epidermidis, S. simulans and S. sciuri. ClyS was active notonly against S. epidermidis including the biofilm-forming strain RP62Abut was also active against S. simulans and S. sciuri suggesting thatClyS recognizes an epitope in the cell wall that is present in allstaphylococcal cells. Set III consisted of a mix of Gram-positive andGram-negative bacteria including representatives of group A, B, C and Estreptococci, oral streptococcal species including S. gordonii, and S.salivarius, as well as S. uberis, Bacillus cereus, Pseudomonasaeruginosa and E. coli. ClyS exhibited no activity against any of theseorganisms.

In another embodiment, a chimeric peptide comprises an isolatedpolypeptide comprising an endopeptidase domain of the S. aureus Twortlysin upstream of the lyphostaphin CWT domain. One example of such alysin is provided in SEQ ID NO:3 (AD119).

In another embodiment, lytic compositions may comprise a mixture of twoor more lysins. The mixture may include a first polypeptide and a secondpeptide where one or both of the polypeptides may lack a desired levelof lytic activity, but the mixture provides desirably specific andeffective lytic activity against a bacteria of interest. For example, acomposition may include an isolated first polypeptide comprising anendopeptidase domain of the S. aureus Twort lysin upstream of thelyphostaphin CWT domain combined with a second isolated polypeptidecomprising an S. epidermidis autolysin amidase domain upstream of thelysostaphin CWT domain. One example of such a composition comprises amixture of SEQ ID NO:3 (AD119) and SEQ ID NO:4 (AD112).

In some examples, the present disclosure pertains to lytic enzymes as aprophylactic treatment for preventing infection those who have possiblybeen exposed to S. aureus bacteria, or as a therapeutic treatment forthose who have already become ill from the infection. The phageassociated lytic enzymes described herein are specific for S. aureusbacteria and preferably effectively and efficiently break down the cellwall of the S. aureus bacteria.

The chimeric lytic enzyme polypeptides described herein may also beemployed as a therapeutic agent. The lytic enzyme polypeptides of thepresent invention can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby the lytic enzymeproduct hereof is combined in admixture with a pharmaceuticallyacceptable carrier vehicle. Compositions which may be used for theprophylactic and therapeutic treatment of a S. aureus bacteria infectionalso includes the shuffled and/or chimeric enzyme and a means ofapplication (such as a carrier system or an oral delivery mode) to themucosal lining of the oral and nasal cavity, such that the enzyme is putin the carrier system or oral delivery mode to reach the mucosa lining.

In one preferred embodiment, a Staphylococcus chimeric lysin, such as alysin of SEQ ID NO:2 (ClyS), is administered as an antibacterialcomposition in combination with a suitable pharmaceutical carrier. Incertain embodiments, the amount of the chimeric bacteriophase lysinpresent is a therapeutically effective amount. “Carriers” as used hereininclude pharmaceutically acceptable carriers, excipients, or stabilizerswhich are nontoxic to the cell or mammal being exposed thereto at thedosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptide;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.These antimicrobial/pharmaceutical compositions may be administeredlocally or systemically.

Routes of administration include topical, ocular, nasal, pulmonary,buccal, parenteral (intravenous, subcutaneous, and intramuscular), oral,parenteral, vaginal and rectal. Also administration from implants ispossible. The compounds of the invention may also be administeredtopically to the skin or mucosa, that is, dermally or transdermally.Typical formulations for this purpose include gels, hydrogels, lotions,solutions, creams, ointments, dusting powders, dressings, foams, films,skin patches, wafers, implants, sponges, fibres, bandages andmicroemulsions. Liposomes may also be used. Typical carriers includealcohol, water, mineral oil, liquid petrolatum, white petrolatum,glycerin, polyethylene glycol and propylene glycol. Penetrationenhancers may be incorporated [see, for example, J Pharm Sci, 88 (10),955-958 by Finnin and Morgan (October 1999).]

The compounds of the invention may also be administered directly intothe blood stream, into muscle, or into an internal organ. Suitable meansfor parenteral administration include intravenous, intraarterial,intraperitoneal, intrathecal, intraventricular, intraurethral,intrasternal, intracranial, intramuscular and subcutaneous. Suitabledevices for parenteral administration include needle (includingmicroneedle) injectors, needle-free injectors and infusion techniques.The compounds of the invention may also be administered intranasally ororally by inhalation, typically in the form of a aerosol.

Suitable antimicrobial preparation forms are, for example granules,powders, tablets, coated tablets, (micro) capsules, suppositories,syrups, emulsions, microemulsions, defined as optically isotropicthermodynamically stable systems consisting of water, oil andsurfactant, liquid crystalline phases, defined as systems characterizedby long-range order but short-range disorder (examples include lamellar,hexagonal and cubic phases, either water- or oil continuous), or theirdispersed counterparts, gels, ointments, dispersions, suspensions,creams, aerosols, droplets or injectable solution in ampule form andalso preparations with protracted release of active compounds, in whosepreparation excipients, diluents, adjuvants or carriers are customarilyused as described above. The pharmaceutical composition may also beprovided in bandages or in sutures or the like.

Many orthopedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis. Late deep infection byS. aureus is a serious complication sometimes leading to loss of theprosthetic joint and is accompanied by significant morbidity andmortality. It may therefore be possible to extend the use of thechimeric bacteriophage lysin described herein (e.g., SEQ ID NO:2) as areplacement for or for use in combination with prophylactic antibioticsin this situation. The chimeric bacteriophage lysin may be administeredby injection with a suitable carrier directly to the site of theorthopedic device in situ to clear the infection, or on a surface of thedevice prior to implantantation. Other injection routes, such assubcutaneous, intramuscular, or intraperitoneal, can be used.Alternative means for administration include transmucosal andtransdermal administration using penetrants such as bile salts orfusidic acids or other detergents. In addition, if a polypeptide orother compounds of the present invention can be formulated in an entericor an encapsulated formulation, oral administration may also bepossible. Administration of these compounds may also be topical and/orlocalized, in the form of salves, pastes, gels, and the like.

Prior to, or at the time the enzyme is put in the carrier system or oraldelivery mode, it may be desirable for a chimeric peptide describedherein to be administered or formulated in a stabilizing bufferenvironment, maintaining a pH range between about 5.0 and about 7.5.Prior to, or at the time the chimeric peptide is put in the carriersystem or oral delivery mode, the enzyme may be in a stabilizing bufferenvironment for maintaining a suitable pH range, such as between about5.0 and about 8.0, including a pH of about 5.0, 6.0, 7.0, 8.0 or any pHinterval of 0.05 therebetween, or any interval that is a multiple of0.05 therebetween, including pH values of 5.2, 6.5, 7.4, 7.5 and 8.5.

There are a number of advantages to using lytic enzymes to treatbacterial infections. The modular design of lysins, with their distinctcatalytic and binding domains, makes them ideal for domain swappingexperiments in which bacterial specificities and catalytic activitiescan be improved or adapted for use against alternate pathogens. Sincethe catalytic and binding targets of lysins (peptidoglycan andassociated carbohydrates, respectively) are largely essential forviability, lysin resistance will be rare.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

The formulations to be used for in vivo administration are preferablysterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. Therapeutic compositions herein generally are placedinto a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems. When treating abacterial exposure or infection, the lytic enzyme may be administered inany suitable fashion, including parenterally or through the oral ornasal cavity.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician.Animal-experiments provide reliable guidance for the determination ofeffective doses for human therapy. Interspecies scaling of effectivedoses can be performed following the principles laid down by Mordenti,J. and Chappell, W. “The use of interspecies scaling in toxicokinetics”In Toxicokinetics and New Drug Development, Yacobi et al., Eds.,Pergamon Press, New York 1989, pp. 42-96.

When in vivo administration of a chimeic peptide lysin is employed,normal dosage amounts may vary from about 10 ng/kg to up to 1000 mg/kgof mammal body weight or more per day, or about 1 μg/kg/day to 10000mg/kg/day, depending upon the route of administration. Guidance as toparticular dosages and methods of delivery is also provided below, aswell as in the literature. It is anticipated that different formulationswill be effective for different treatment compounds and differentdisorders, that administration targeting one organ or tissue, forexample, may necessitate delivery in a manner different from that toanother organ or tissue.

The effective dosage rates or amounts of the chimeric peptide to beadministered parenterally, and the duration of treatment will depend inpart on the seriousness of the infection, the weight of the patient, theduration of exposure of the recipient to the infectious bacteria, theseriousness of the infection, and a variety of a number of othervariables. The composition may be applied anywhere from once to severaltimes a day, and may be applied for a short or long term period. Theusage may last for days or weeks. Any dosage form employed shouldprovide for a minimum number of units for a minimum amount of time. Theconcentration of the active units of a chimeric peptide believed toprovide for an effective amount or dosage of enzyme may be in the rangeof about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 units/ml up to about10,000,000 units/ml of composition, in a range of about 1000 units/ml toabout 10,000,000 units/ml, and from about 10,000 to 10,000,000 units/ml.

Additionally, a number of methods can be used to assist in transportingthe enzyme across the cell membrane. The enzyme can be transported in aliposome, with the enzyme be “inserted” in the liposomes by knowntechniques. Similarly, the enzyme may be in a reverse micelle. Theenzyme can also be pegylated, attaching the polyethylene glycol to thenon-active part of the enzyme. Alternatively, hydrophobic molecules canbe used to transport the enzyme across the cell membrane. Finally, theglycosylation of the enzyme can be used to target specificinternalization receptors on the membrane of the cell.

Another preferred embodiment provides for a composition comprising aStaphylococcus chimeric lysin bacterial binding protein such as a lysinof SEQ ID NO:2 (ClyS), with other lytic enzymes which are useful forsanitizing or decontaminating porous surfaces e.g. textiles, carpeting.Furthermore, the composition of lytic enzymes may be used todecontaminate veterinarian surgical or examination areas, where suchareas may be thought to harbor infectious organisms susceptible to thebacteriostatic or bacteriocidal activity.

In a further preferred embodiment, a Staphylococcus chimeric lysin suchas a lysin of SEQ ID NO:2 (ClyS) may be combined with otherbacteriostatic or bacteriocidal agents useful for decontamination ofinanimate solid surfaces suspected of containing infectious bacteria, orfor decontamination of porous surfaces.

EXAMPLES Example 1: Identification of Specific Binding Peptides andDevelopment of Chimeric Lysins

We conducted conserved domain searches of Staphylococcus-specific phageand prophage lysin protein sequences in the National Center forBiotechnology Information database. The lysins were classified based onhomology to known domains in the database. We identified several lysinsincluding the S. aureus phage phiNM3 lysin (protein accession numberYP_908849), S. aureus prophage phi13 amidase (accession numberNP_803402), S. aureus prophage MW2 amidase (accession numberNP_646703.1), etc. that shared 100% sequence identity with each otherand had a conserved CHAP domain within their catalytic domain. However,the C-terminal domain of these lysins did not display homology to anyknown domains in the database (FIG. 1).

Since the attempts to express a native staphylococcal lysin wereunsuccessful, we decided to develop chimeric lysins by taking advantageof the modular nature of lysins. Traditionally, Bacillus-specific lysinsare expressed at high levels and are soluble in E. coli. Therefore, ourfirst attempt was to engineer a 16-amino acid peptide (4) that isconserved in several S. aureus-specific lysins (Lu J Z, Fujiwara T,Komatsuzawa H, Sugai M, Sakon J. 2006. Cell wall-targeting domain ofglycylglycine endopeptidase distinguishes among peptidoglycancross-bridges. (Lue et al. (2006) J. Biol. Chem. 281(1):549-58) Thecatalytic domain of the Bacillus-specific lysin PlyB was used togenerate chimera AD 103 (FIG. 4) (SEQ ID NO: 13). The chimeras weretested for expression, solubility and activity. Then the entireC-terminal CWT domain of PlyB (6) was replaced by the putativeC-terminal domain of S. aureus phage Twort lysin (8) to obtain AD 105(SEQ ID NO:14). This chimera was not active and so we engineered thelysostaphin CWT domain (10) downstream of PlyB-catalytic domain (2) toget AD 107 (SEQ ID NO:15). Although this chimera had expression, thesolubility was poor and there was no activity. The next step was toengineer a S. epidermidis autolysin amidase domain (12) upstream of thelysostaphin CWT domain (10) which resulted in AD 112 (SEQ ID NO:4). AD112 expressed very well and the protein was also very soluble but therewas no lytic activity. However, we observed that the S. aureus cellsclumped when exposed to AD 112. Since the lysostaphin catalytic domain(an amidase) (10) in AD 112 was of bacterial origin, we attempted toengineer a phage-derived catalytic domain upstream of the lysostaphinCWT. For this, the endopeptidase domain in Twort lysin (14) was used toconstruct chimera AD 119 (SEQ ID NO:3). We observed poor expression forAD 119 but the chimera was soluble. Although in our lytic assays AD 119alone did not show significant activity, when combined with chimera AD112 the activity was significantly enhanced. Since we identified phiNM3CWT domain from our conserved domains searches and observed that phiNM3CWT exhibited Staphylococcus-specific binding, we constructed chimerasAD 126 (SEQ ID NO:16) and AD 127 (SEQ ID NO:2) by engineering S.epidermidis autolysin amidase (12) and Twort lysin endopeptidase (14)domains upstream of phiNM3 CWT domain (2) (SEQ ID NO:1), respectively.Chimera AD 126 had no expression or activity but AD 127 was soluble andhad very high activity but low expression (FIG. 4). To overcome lowexpression of AD 127 construct, the entire chimera gene was cloned intoexpression vector pJML6 to generate pAD 138. The expression, solubilityand activity of AD 127 from the pAD138 construct was very high.Therefore, this chimera was named ‘ClyS’ for Chimeric lysin forStaphylococcus. The amino acid sequence for ClyS (i.e., SEQ ID NO:2) isprovided in FIG. 5A.

Example 2: Construction of the ClyS Chimeric Lysin

Bacterial strains (Table 1) were stored at −80° C. routinely grown at37° C. Staphylococcal strains used in this study were grown inTrypticase Soy Broth (TSB) media, streptococcal strains were grown inTHY (Todd-Hewitt broth, 1% wt/vol yeast extract) media, B. cereus and P.aeruginosa were grown in BHI (Brain Heart Infusion) media while E. coliwas cultivated in LB (Luria Bertani) media.

The chimeric lysin was constructed by amplifying and ligating individualdomains from respective genes. For this, the Twort endopeptidase domainwas PCR amplified from plasmid pCR2.1 plyTW which contains the entirelysin (plyTW) gene using primers TW-Endo-NcoI-F:5′-CTAGCCATGGAAACCCTGAAACAAGCAG-3′ (SEQ ID NO:5) and TW-Endo-PstI-R:5′-ACATGCTGCAGAACCATATTGTAATTAATATTAGTTCTATC-3′(SEQ ID NO:6). The cellwall targeting (CWT) domain was PCR amplified from S. aureus strain 8325genomic DNA using primers NM3-CBD-PstI-F:5′-ACATGCTGCAGGGTAAATCTGCAAGTAAAATAACAG-3′ (SEQ ID NO:7) andNM3-CBD-Hind-R: 5′-CCCAAGCTTAAAACACTTCTTTCACAATCAATCTC-3′(SEQ ID NO:8).The two PCR amplicons were ligated using the PstI restrictionendonuclease site. The ligated product was cloned into pBAD24 vectorusing the NcoI-HindIII cloning sites to generate recombinant plasmidpAD127. In the second step, the entire DNA fragment corresponding toclyS was PCR amplified from pAD124 using primers NM3-Lys-Xba-F:5′-CTAGTCTAGAGGTGGAATAATGAAAACATACAGTGAAGCAAG-3′ (SEQ ID NO:9) andprimer NM3-CBD-Hind-R(SEQ ID NO:8). The PCR product was cloned intoexpression vector pJML6 to generate pAD138. The sequence of ClyS wasconfirmed by sequencing. The recombinant plasmid pAD138 was transformedinto E. coli DH5a cells.

Example 3: Overexpression and Purification of ClyS

ClyS was induced overnight from E. coli DH5a (pAD138) cells with lactose(10 g/500 ml final concentration) at 30° C. Cells were harvested bycentrifugation, resuspended in buffer A (20 mM phosphate buffer (PB), 1mM DTT (dithiothreitol)) and lysed by an EmulsiFlex-05 high pressurehomogenizer (Avestin) at 400 C. The lysates were cleared bycentrifugation (2×50,000×g) for 30 minutes at 4° C. and the supernatantapplied to a CM-sepharose column (Amersham Pharmacia, Piscataway, N.J.).ClyS was eluted with buffer A+1M NaCl using a linear gradient of 0-50% Bin 15 columns volumes. Fractions were analyzed for lytic activity aspreviously described (Daniel et al, 2001). Fractions displaying lyticactivity were pooled and dialyzed overnight against buffer B (PB, 1 mMDTT, 50 mM NaCl). The dialyzed sample was applied to a hydroxylapatite(MacroPrep TypeII 40 μm, BioRad) column and eluted with elution buffer(500 mM PB+50 mM NaCl+1 mM DTT) using a linear gradient of 0-100% B in20 columns volumes. The fractions were analyzed by SDS-PAGE and forlytic activity. Active clean fractions of ClyS were pooled and dialyzedagainst buffer B. Protein concentration was determined with the BCAmethod (Sigma, St. Louis, Mo.).

Example 4: Quantification of ClyS Activity

ClyS activity was measured as previously described (Daniel et al, 2001),with some modifications. Briefly, S. aureus strain 8325-4 was grown toan OD₆₀₀ of 0.25-0.3, centrifuged, and resuspended in PB to a finalOD₆₀₀ of 0.8-1.0. Two-fold serial dilutions of purified ClyS (100 μl)were added to 100 μl of bacterial suspension in 96-well plates (Costar)and the decrease in OD600 was monitored by a Spectramax Plus 384spectrophotometer (Molecular Devices) over 30 min at 37° C. ClySactivity in units per milliliter was defined as the reciprocal of thehighest dilution of lysin that decreased the absorbance by 50% in 15minutes.

Example 5: Measuring In Vitro ClyS Activity

The viability assay of ClyS was tested as previously described (Nelsonet al, 2001). Briefly, logphase cultures of S. aureus strain 8325-4 wereresuspended in PB to OD₆₀₀ of 0.8-1.0. 50U of ClyS or the correspondingvolume of PB was added to bacterial cells and aliquots were removed,serially diluted, and plated at 1, 5, 10, 30, and 60 minutes to assessthe viability of the treated and control cells. All experiments wereperformed in triplicate. The activity of ClyS on various bacterialstrains was tested as described previously (Schuch et al, 2002).Briefly, logphase bacterial cells were treated with 50U of ClyS at 37°C. for 15 minutes. The samples were serially diluted and plated. Controlexperiments with the addition of phosphate buffer (pH 7.0) wereperformed under the same conditions.

Example 6: Measuring ClyS Activity as a Function of pH and Salt Profile

The effect of pH on ClyS activity was determined as previously describedusing the universal buffer system pH 4-10 (Yoong et al). Briefly,logphase 8325-4 cells were resuspended in the universal buffer systemand incubated with 50U of ClyS for 15 minutes. The final pH of eachreaction was checked by pH paper. The samples were serially diluted andplated. In controls, PB replaced ClyS.

Similarly the effect of salt concentration on the lytic activity of ClySwas determined by incubating 50U of ClyS with logphase 8325-4 cells inPB containing NaCl at a final concentration of 25-500 mM for 15 minutes.The samples were serially diluted and plated to determine the viabilitycounts.

Example 7: Microscopy of ClyS

S. aureus strain 8325-4 was grown to log-phase, centrifuged andresuspended in PBS to an absorbance at 600 nm of 1.0. The bacterialsuspension was incubated with 50U of ClyS at room temperature. The lyticreaction was terminated after 1 minute and 5 minutes by addingglutaraldehyde (final concentration 2.5%). The suspension was pelletedby centrifugation and overlaid with 2.5% glutaraldehyde in 0.1 Mcacodylate buffer (pH 7.4). The samples were then postfixed in 1% osmiumtetroxide, block stained with uranyl acetate and processed according tostandard procedures by The Rockefeller University Electron MicroscopyService.

Flourescent labeling and binding analyses were performed on phiNM3 CWT.S. aureus strain 8325-4 genomic DNA was used to amplify the putative CWTof phiNM3 lysin using primers NM3-FWD5′-CATGCCATGGGTAAATCTGCAAGTAAAATAACAG-3′ (SEQ ID NO:10) and NM3-REV5′-CCCAAGCTTAAAACACTTCTTTCACAATCAATCTC-3′(SEQ ID NO:11). The resultingamplicon was cloned into the arabinose-inducible expression vectorpBAD24. Positive clones containing the insert were confirmed bysequencing. The approximately 10-kDa phiNM3 CWT protein was expressedand the protein was purified in one step by cation-exhangechromatography. The purified protein (1 mg/ml) was incubated with 10 μlof FITC (1 mg/ml) for 1 hour. Excess FITC was removed on a desaltingcolumn. The labeled-protein (50 μg) was incubated with bacterial cellsfor 10 minutes, washed 3× with phosphate-buffered saline (pH 7.4) andobserved under fluorescence microscope.

Example 8: Measuring In Vivo Activity of ClyS

MRSA strain would be grown to log-phase, centrifuged and resuspended toa predefined titer of about 1010 cfu/ml. For intranasal infection,6-wk-old female C57BL/6J, outbread Swiss or BALB/c mice (weight range 22to 24 g, Charles River Laboratories, Wilmington, Mass.) would beanesthetized with a mixture of ketamine (Fort Dodge Animal Health, FortDodge, Iowa, 1.2 mg/animal) and xylazine (Miles Inc., Shawnee Mission,Kans., 0.25 mg/animal), and inoculated with 15 μl of the bacterialsuspension per nostril (n=10). The animals would be divided into 2groups and administered various concentrations of ClyS or sterile salineintraperitoneally six hours after infection and every six hoursthereafter for 3 days. The survival rate for each group would beobserved up to 7 days post infection. For intraperitoneal infection,mice would be infected intraperitoneally with 100 μl of the bacterialsuspension (n=10). The animals would be divided into 2 groups andadministered various concentrations of ClyS or sterile salineintraperitoneally six hours after infection and every six hoursthereafter for 3 days. The survival rate for each group would beobserved up to 7 days post infection.

Example 9. The Linker Region by Itself does not Confer Solubility to aChimera

Since the ClyS construct was the only chimera that was highly solubleand active against staphylococci, we hypothesized that the linker regioncomprising of amino acid residues 142 through 185 of ClyS may be crucialfor solubility. We had previously cloned and expressed the native phiNM3lysin and observed that the protein was insoluble. To test thishypothesis, we replaced the endopeptidase domain of ClyS with theamidase domain of phiNM3 lysin upstream of the linker region of ClyS(ami-link-ClyS) and expressed the chimera (data not shown). We observedthat similar to the native phiNM3 lysin, the ami-link-ClyS chimera wasinsoluble and expressed as inclusion bodies. We also tested the lysatesof ami-link-ClyS for activity against staphylococci and did not observeany lytic activity confirming that the protein was insoluble andtherefore inactive. Thus, it is the unique combination of the N and Cterminal domains that are the subject of this patent that allow for asoluble complex to occur and behave as described herein.

Example 10. In Vivo Nasal Decolonization of MRSA by ClyS

Carriage of both MSSA and MRSA in the human anterior nares is the majorreservoir for S. aureus infection. Studies have shown that roughly 80%of the population could be nasally colonized by S. aureus, and thatcolonization can be an increased risk factor for developing other moreserious S. aureus infections (Kluytmans, J., A. van Belkum. 1997. Nasalcarriage of Staphylococcus aureus: epidemiology, underlying mechanisms,and associated risks. Clin Microbiol Rev 10(3): 505-20.). Elimination ofnasal carriage in the community or in the hospital setting thus couldpossibly reduce the risk of infection and slow the spread of drugresistant S. aureus (Kluytmans et al. (1997)). To study the potential ofClyS to reduce MRSA colonization of the nasal mucosa, C57BL/6J mice wereintranasally inoculated with ˜2×10⁷ of a spontaneously streptomycinresistant strain of MRSA (191-SM^(R)). Twenty-four hours post-infectionmice were administered three doses hourly of either phosphate bufferedsaline (control) or ClyS (960 μg) into the nasal passages. One hourafter the last treatment, mice were sacrificed and bacteria colonieswere enumerated on Spectra MRSA agar, (a selective chromogenic mediumdeveloped to diagnostically detect MRSA nasal colonization) and Columbiablood agar. No significant differences in CFU were obtained betweenplating to Spectra MRSA agar or Columbia blood agar (Data not shown)Three independent experiments were performed to evaluate a total 20 micefor each treatment group (FIG. 11). Compared to the buffer alone control(Avg. 12,273 CFU/cavity), ClyS treatment (Avg. 1198 CFU/cavity)significantly (p<0.001) reduced the mean CFU on the nasal mucosa.

Example 11. ClyS Treatment of Systemic MRSA Infections

In order to assess whether ClyS treatment could prevent death resultingfrom systemic MRSA infections, 4 week old FVB/NJ mice wereintraperitonally injected with 5×10⁵ CFU of the community-acquired MRSAstrain MW2 in 5% mucin. Preliminary experiments determined that 5×10⁵CFU was 10× the LD₁₀₀ dose for a twenty-four hour period. Furthermore,within 3 hours of IP injection the MRSA infection was systemic, i.e.,MRSA were recovered in high numbers from heart, liver, spleen, andkidney (data not shown). Treatment occurred three hours post-infection,with either 20 mM phosphate buffer or 1 mg of ClyS in 20 mM phosphatebuffer injected IP (intraperitoneally). Mice were then monitored forsurvival over ten days. The results from three independent experimentswere combined (ClyS treatment, n=16; buffer treatment, n=14) and themice survival data plotted with a Kaplan Meier Survival curve (FIG. 12).Within twenty-four hours all of the control mice died of bacterialsepsis, while only 2/16 of ClyS treated mice died at forty-eight hours,and the remaining mice (14/16, 88%) survived over the course of theexperiments (FIG. 12).

Example 12. ClyS Showed Synergistic Interaction with Vancomycin andOxacillin

We used the checkerboard broth-microdilution assay to test theinteraction of ClyS with vancomycin and with oxacillin. The vancomycinMIC for VISA strain Mu50 was 8 μg/ml and the oxacillin MIC for MRSAstrain COL was 32 μg/ml, while the ClyS MIC was 6 and 8 U/ml for bothstrains tested (Mu50 and COL respectively). Isobolograms for ClyS withvancomycin and ClyS with oxacillin was plotted by transcribing theenzyme concentrations along the inhibitory line on the microtiter platein an x/y plot. The shape of the curves for both interactions werecharacteristic of a synergistic interaction (FIG. 13) and were furtherconfirmed by calculating the ΣFICI for both interactions which was ≤0.5.

Example 13. In Vivo Synergy of Oxacillin and ClyS in the Treatment ofSystemic MRSA Infections

In vitro experiments showed that ClyS acted synergistically withoxacillin (FIG. 14). To determine if this effect could be seen in oursystemic MRSA infection model, FVB/NJ mice were intraperitonallyinjected with ˜5×10⁵ CFUs of the MRSA strain MW2 as above. Three hourspost infection mice were treated in parallel, with a lower IP dose of130 μg/mouse of ClyS combined with different concentrations of oxacillin(10-100 μg/mouse) or buffer alone controls. Preliminary experimentsdetermined that an ED₃₀ dose of ClyS (130 μg/mouse) had minimal efficacyto evaluate the effect of combinatorial treatment with oxacillin (datanot shown). Mice were monitored for survival for 10 days and the resultsof 5 independent experiments were combined and plotted in a Kaplan MeierSurvival curve (FIG. 14). While only 30% (6/20 alive) to 35% (8/23alive) of mice survived with individual treatments of either 130μg/mouse of ClyS or 100 μg/mouse of oxacillin, respectively, neitherdiffered significantly from the survival rate of the buffer alonecontrol, 13% (2/15 alive). Conversely, a single dose of the combinedtreatment of intraperitoneal injected ClyS (130 μg) with either 0 μg or50 μg of intramuscular injected oxacillin significantly increased mousesurvival (80%, 8/10 alive; 82%, 18/22 alive respectively) compared tothe individual treatments and buffer alone (FIG. 14).

Example 16

Modification of ClyS. The G¹⁶⁶ residue of ClyS (SEQ ID NO:2) was changedto a proline by site directed mutagenesis (creating pClyS). When thepurified pClyS molecule (SEQ ID NO: 17) was subjected to stabilitystudies at 21° C. for 5 days, the pClyS was found to be significantlymore stable in the presence of 5 mM DTT the native ClyS with or withoutDTT (FIG. 15).

While the invention has been described and illustrated herein byreference to various specific materials, procedures, and examples, it isunderstood that the invention is not restricted to the particularmaterials, combinations of materials, and procedures selected for thatpurpose. Numerous variations of such details can be implied and will beappreciated by those skilled in the art.

1. A chimeric bacteriophage lysin comprising a catalytic domain of a first Staphylococcus-specific phage lysin and a binding domain of a second Staphylococcus-specific phage lysin, wherein the chimeric lysin has killing activity against Staphylococcus aureus.
 2. The chimeric lysin of claim 1, wherein the binding domain is not an SH3b-like cell wall targeting domain.
 3. The chimeric lysin of claim 1, wherein the catalytic domain is bound to the N-terminal end of the binding domain.
 4. The chimeric lysin of claim 1, wherein the binding domain comprises the polypeptide sequence of SEQ ID NO:1, or a variant thereof wherein said variant is capable of binding specifically to staphylococci.
 5. The chimeric lysin of claim 1, wherein the catalytic domain is the Twort endopeptidase domain.
 6. The chimeric lysin of claim 1, wherein the chimeric lysin comprises the polypeptide sequence of SEQ ID NO:2, or a variant thereof wherein said variant has killing activity against Staphylococcus aureus.
 7. (canceled)
 8. The chimeric lysin of claim 1, wherein the chimeric lysin has killing activity against methicillin-resistant and methicillin-sensitive strains of S. aureus.
 9. (canceled)
 10. The chimeric lysin of claim 1, wherein the chimeric lysin kills Staphylococcus epidermidis.
 11. The chimeric lysin of claim 1 having killing activity against Staphylococcal bacteria over a pH range of about 4-10.
 12. The chimeric lysin of claim 1, wherein the lysin has a solubility of at least about 3 mg/mL in phosphate buffered saline (PBS).
 13. A method of treating a mammal with a staphylococcal infection comprising administering to said mammal a chimeric bacteriophage lysin of claim
 1. 14. A method of preventing infection of a mammal colonized by staphylococci comprising administering to said mammal a chimeric bacteriophage lysin of claim
 1. 15. A method of detecting the presence of a staphylococcal bacteria comprising the step of contacting a peptide comprising the amino acid sequence of SEQ ID NO:1 that specifically binds to staphylococcal bacteria, or comprising a variant of SEQ ID NO: 1 wherein the variant specifically binds to staphylococcal bacteria, with a sample, and detecting the peptide within the sample.
 16. The method of claim 15, where the peptide is a GFP fluorescent reporting fusion protein.
 17. The method of claim 13, wherein the chimeric lysin has a solubility of at least about 1 mg/ml in phosphate buffered saline.
 18. The method of claim 13, wherein the chimeric lysin selectively binds to staphylococcal bacteria in the presence of one or more additional bacteria selected from the group consisting of: E coli, B. cereus and P. aeruginosa.
 19. The method of claim 13, where the chimeric lysin is a soluble chimeric bacteriophage lysin comprising a catalytic domain of a first Staphylococcus-specific phage lysin and the binding domain of SEQ ID NO:1.
 20. The method of claim 13, wherein the chimeric lysin comprises the amino acid sequence of SEQ ID NO:2, or a variant thereof wherein said variant has killing activity against Staphylococcus aureus.
 21. (canceled)
 22. An anti-microbial composition for sanitizing or decontaminating porous or non-porous surfaces comprising a chimeric bacteriophage lysin of claim
 1. 23. A method for decontaminating inanimate surfaces suspected of containing infectious bacteria comprising treatment of said surfaces with a bacteriocidal or bacteriostatically effective amount of the composition of claim
 22. 